The proteome is defined as the array of proteins expressed in a cell, tissue or organ at a particular time, under a particular set of conditions. This set of proteins is responsible for cellular structure and function. Disruptions in the protein machinery contribute to uncontrolled cell growth and formation of a tumor. Proteome science (proteomics) employs a combination of sophisticated separation techniques, mass spectrometry and bioinformatics for the investigation of proteomes. In the proposed research program, the proteomics approach will be applied to the examination of the proteome in malignant prostate tumors. Specific aim 1: to develop a comprehensive proteomics-based methodology for the separation and characterization of human prostate proteins. The proteomics protocol will be improved and optimized to achieve maximum efficiency and success rate in the solubilization, separation, detection and characterization of prostate proteins. The methodology will incorporate cutting-edge technological advances in tissue preparation, two-dimensional gel electrophoresis and protein identification. The use of Laser Capture Microdissection for the procurement of prostate cells of a specific type will be tested. Performance (sensitivity, throughput, quality of data output) of two state-of-the-art mass spectrometric techniques will be evaluated. Specific aim 2: to use the proteomics approach to examine protein expression in normal and malignant prostate. The prostate proteins from normal or cancer tissue will be separated by two-dimensional gel electrophoresis, and the protein expression profiles from normal vs. tumor prostate will be compared. The proteins whose abundances are altered in prostate cancer will be identified and characterized by mass spectrometry and searching of protein or DNA sequence databases. The proposed study will provide a robust, high-throughput methodology for the molecular characterization of the human prostate, and it will yield pilot data concerning cancer-related changes in the prostate proteome. In the long run, knowledge of prostate cancer characteristics at the protein level will bring more understanding of the mechanisms of prostate tumorigenesis; it will pinpoint biomarkers that may be used for the detection and diagnosis of prostate cancer; and it will identify possible targets for drug intervention.